DNA interstrand crosslinks are a particularly lethal form of DNA damage that represent an absolute block to replication and transcription. Chemicals forming crosslinks have proven to be highly toxic when found in nature, uniquely potent as chemotherapeutics in specific cancers, and effective treatments for a range of diseases states involving hyperplastic or displastic conditions. Although several genes have been isolated that, when mutated, render cells hypersensitive to crosslinks, many aspects of how these complex lesions are repaired and processed in cells remain unknown. Much of what we know about interstrand crosslink repair has come from eukaryotic studies in extracts and suggest that both replication-dependent and replication-independent mechanisms exist. Both mechanisms are proposed to involve multiple repair pathways, coupling components of nucleotide excision repair with recombination, translesion synthesis, as well as other alternative nuclease complexes. However, after the initial incision event, all these models remain highly speculative, and are hampered by the challenges of reconstituting this multi-step, multi-pathway repair process as well as by the complexity and lack of cellular assays available in mammalian cells. Here, we propose to directly identify the cellular pathways and structural intermediates that arise during the repair of interstrand crosslinks in vivo using the model organism of E.coli, where the processes of replication and repair are highly conserved. In E. coli, we have established unique cellular assays to monitor the replication fork processing and global repair for these lesions. In addition, in preliminary data, we show that we have identified an alternative endonuclease, similar to mammalian cells, that couples with the nucleotide excision repair complex and is important for crosslink repair. These assays will allow us to directly and definitively identify the repair and progressive intermediates that arise during crosslink repair i vivo We describe three aims that will be accomplished. 1) We will identify the genes involved in repairing DNA interstrand crosslinks in E. coli and determine whether they operate in a replication-dependent or replication-independent (global genomic) repair pathway in vivo. 2) We will identify the cellular intermediates and biochemical pathway associated with the replication-independent repair of DNA crosslinks in vivo. 3) We will identify the cellular intermediates and biochemical pathway associated with the replication-dependent repair of DNA crosslinks in vivo. The results of these studies will identify the pathways operating in the repair of this medically relevant lesion in vivo and are likely to suggest novel therapeutic approaches that utilize these lesions in the treatment of cancer and other hyperproliferative diseases.